Biochemical, chemical, physical and biological characteristics of drug and exploration of novel drugs from natural sourcesare studied in field of pharmacognosy.1 The subjects of Botany, Chemistry and Pharmacology are involved in the study of medications from plant sources.2 Pharmacognostical research consists of phytochemistry, microbiology, biosynthesis, biotransformations, chemotaxonomy and other biological and chemical sciences.3 Measure of the strength or potency of the functional constituents or indicator compound is known as standardization. It’s used to mention the batch-to-batch consistency of effective ingredients instead of the intensity and reliability of the pharmacological effects and within the industry and several regulatory agencies has been the matter of extreme discussion.4
Pharmacognosist Professors Jerry L. McLaughlin and colleagues established simple brine shrimp lethality ``bench top’’ bioassay,5 that proved suif to regulate the diagnosing of anti-tumours and cytotoxics compound from plant.6 Phytotoxic activity is the initial weedicide bioactivity of natural products for screening potential phytotoxic plants.7A variety of novel antimicrobial drugs in the recent period have been developed by pharmaceutical industries but at the same time opposition to these drugs by microbes has built up. Generally, bacteria exhibit the genetic capability to transfer and develop resistance to drugs used as healing agents.8 Use of synthetic fungicides for control of fungal infestations is common, but its use is constrained because of the noxious effects on organism’s healthiness and on surroundings.9
Oxalis pes-caprae L.is perennial, herbaceous plant withbulbiferous andunderground stem.It is native to South Africa. It is naturalized to West Europe and Mediterranean region, South West Asia, North Africa, Turkey and Iran. In the plains having shady places, it is found as a weed.10 The plant is edible and in moderate quantity is unhazardous. This plant also a traditional constituent in recipe i.e. water blommet jiebredie (water flowers stew) in South Africa.11 In several traditions it is used in folk medicines, as foods and for oxalic acid. For treating tapeworm and may be other worms, raw bulbs are used. It is also utilized as diuretic. Fleshy underground runners are also eaten with milk.12 Yellow dye is obtained from the golden petals.13 The roots, stems and leaves of Oxalis pes-caprae have high medicinal values and have the function of anti-inflammatory and analgesia, clearing heat, removing toxicity.14
MATERIALS AND METHODS
Plant Collection and Preservation
Fresh collection of Oxalis pes-caprae at their flowering stagewas carried out from Department of Botany, University of Peshawar, Pakistan. Plant materials were cleaned, washed and garbled. Few fresh plants of Oxalis pes-caprae were pressed, dried, pasted on herbarium sheet and assigned voucher numbers (Bot. 20128 (PUP) and deposit in herbarium (Department of Botany, University ofPeshawar, Pakistan) for reference. Collection was dried in shade at about 25±3 0C and powdered with electric grinder. For protecting powder frommolds, insects attack andmoisture, these were stored inair tight bottles and used for various biochemicals and biological research studies.
In airtightbottles, 500ml of different solvents (ethanol, methanol, n-hexane, acetone, chloroform) and 100gm powder of plants were mixed and kept for 7 days with occasional shaking. After this extract was filtered and in rotary evaporators filtrates were dried.
For measuring therapeuticpotential of Oxalis pes-caprae the following pharmacological studies were conducted.
Cytotoxic activity was performed for ethanolic, methanolic, acetone, n-hexane and chloroform extractsof Oxalis pes-caprae bybrine shrimp bioassay using methodology of.15
Artemia salina (shrimps eggs), sea salt solution (38g/L of distilledwater), illuminating source (lamp) to attractbrine-shrimp larvae, hatching tray with perforated partition, Pasture’s pipettes, magnifying lens, micro pipettes (5, 50,500μl), test samples, test tube stands, test tubes, ethanol, methanol, acetone, n- hexane and chloroform.
Technique for Hatching
Filtered brinesolution was put inhatching tray (rectangular dish (22x32 cm). 2 unequalpartitions (perforated) were made in tray. 25mg brine shrimp eggs powder was scattered in smaller portion. The larger portion was remaining uncover and smaller was wrap with black paper. For hatching of eggs, tray was kept at room temperature. For illuminating open portion of tray, lamp was hanging over tray. The hatched napualli were viewed to move toward theenlightened portion.
Preparation of Sample
Sample was prepared by dissolving 20mg of extract in 2ml of solvent. To test tubes (3 test tubes /concentration) 5, 50 and 500 μl solution was transferred i.e. equivalent to10, 100 and 1000 μg/ml respectively. Solvents could evaporate overnight and to each test tube 5ml seawater solution (38 g/L) was added and transferred 10 larvae with help of Pasteur pipettes. At room temperature (25-27°C), test tubes were kept under illumination. Test tubes filled with brine solution and reference cytotoxic drug used for negative and positive controls respectively.
Numbers of dead and live napualli were counted after 24 hr. The data was analyzed through Biostat software to determine LD50 values.16
The phytotoxic activity of ethanolic, methanolic, acetone, n-Hexane and chloroform extracts of Oxalis pes-caprae was investigated by applying Lamna minor (as test species) following.15
Distilled water, micropipettes (10-100 μl, 100-1000 μl), flask 250ml, test samples (extracts), Lemna minor (test species), filter paper, oven, petri dishes, luminarflow hood, glass vials, E- medium, brush.
For Lemna minor bioassay, E-medium was made from various mineral nutrients having known quantity (Table 1). First the salts were weighed in required quantity then dissolved in distill water and volume was made up to 1000ml. By addition of KOH, pH was adjustedbetween 5.5-6.00.
Sample was prepared by dissolving 15mg of extract in 1.5ml of solvent and to sterilized petri dish (3 petri dishesfor each concentration) 5, 50 and 500μl solutions were transferred i.e. equivalent to 10, 100 and 1000μg/ml respectively. Solvents could evaporate overnight in sterilized condition in laminar flow. To each petri dish 20ml E.medium was added. Petri dishes filled with E. medium alone and standard drug (Atrazine) used as negative and positive controls respectively. 10 plants of Lemna minor with two fronds were transferred to each petri dish. Numbers of frondswere counted on 3rd and 7th day of experiment. With reference to negative control, percent growth inhibition was noted by applying formula
Results for phytotoxicity were analyzed by using Biostat software to determine FI50 values.16
Anti-bacterial screening was conducted for ethanolic, methanolic, acetone, n- Hexane and chloroform extracts ofOxalis pes-caprae following.17
Nutrient broth, test samples, nutrient agar, Laminar flow unit, Standard antibiotics (streptomycin), micropipettes, sterile cotton swab, petri dishes, test tubes, incubator, autoclave, filter paper discs, DMSO.
Antibacterial activities of the plants were conducted by filter paper disc method as explained by.17 On nutrient broth, bacterial strains were cultured and before experiments incubated for 24 hr. Nutrient agar was melted, cooled to 400C and transfer into sterilized petridishes. By using sterilized cotton swab, 4 to 8 hr old bacterial culture was spread on surface of nutrient agar. Betweeneach streaking, turning the plate 600 repeating the procedure thrice. Filter paper discs were placed in petridishes and to these discs sample extracts, four concentrations i.e. 100ppm, 1000ppm, 1500ppm and 2500ppm were applied. Other discs were filled with DMSO and 100ppm streptomycin used as negative and positive controls respectively. For 24 hr the plates were incubated at 370C and then noted for zones of inhibition.
Seven days old fungal cultures, test samples, potato dextrose agar (PDA), Dimethyle sulphoxide (DMSO), petri dishes, cork borer, micropepites, autoclave, incubator, Laminar flow Unit (LFU), spirit, standard antibiotic (streptomycin), fungicide.
25 ml pre-sterilized PDA was poured in each pre-sterilized petridishes under sterile conditions and allowed to solidify. Wells of 6mm diameter was made by sterile cork borer in media. To the wells, sample extract of two concentrations i.e. 100ppm and 1000ppm was added through micropipette. Next each petri dish was inoculated with 6 mm diameter piece of inoculumcut off from seven days old fungal culture. Media filled with DMSO and fungicide was used for negative and positive controls respectively. Petri dishes were incubated at 270C for7 days. During incubation cultures were examined twice weekly. Growth in media was calculated by measuring growth (mm) of fungal strains loaded with DMSO, fungicide and sample. Percentage inhibition of fungal growth was calculated by following formula.19,20
Where, Gn = Mycelial growth in normal
Gt = Myclial growth in test
In the current work Brine shrimp’s lethality bioassay was conducted for knowing the cytotoxicity of ethanolic, methanolic, acetone, n-hexane and chloroform extractof O. pes-caprae. Three concentrations (10ppm, 100ppm, and 1000ppm) were applied against brine shrimps. The extracts were found to produce dose dependent cytotoxicities, based on the following criteria
30-40% lethality – Low activity
50% lethality – moderate activity
60-70% lethality –good activity
Above 70% lethality – significant activity
The highest significant cytotoxicity was shown by ethanolic extract which caused 100% (LD50 8.98) cytoxicity at 1000ppm followed by chloroform extract (93.33%), n-hexane (90%), methanol and acetone (86.66%). OPE also showed significant effect at 100ppm i.e. 73.33%. While at 10ppm and 100ppm all extracts depicted low to good activity. The result was shown in Table 2 and Figure 1.
In the present work, the phytotoxic potential of crude ethanolic, methanolic, acetone, n-hexane and chloroform extract of O. pes-caprae was evaluated by using Lemna minor. Three phytotoxic doses were used. The extract showed low to moderate level inhibitions of frond proliferation, w. r. t following criteria (given below) used for phytotoxic potential
30-40% inhibition: Low activity
50% inhibition: moderate inhibition
60-70% inhibition: good activity
Above 70% inhibition – significant activity
The effect of extract was dose dependent. The data was taken on fourth day and seventh day of experiment. On fourth day the significant effect was shown by chloroform extract at 1000ppm (76%, FI50 0.073). All other extracts showed low to good activity at 10ppm and 100ppm. On seventh day of experiment the significant inhibition was shown by chloroform extract at 100ppm which was 90% with FI50 0.073. The entire extracts showed moderate to significant activity at 10 and 1000ppm. The results are summarized in Table 3 and 4, Figure 2 and 3.
For curing bacterial infections, anti-microbial activity gives chief basis. In current study ethanolic, methanolic, acetone, n-hexane and chloroform extract of O. pes-caprae against Xanthomonas, Clavibacter machengnitis and Bacillus at 100, 1000, 1500 and 2500ppm were tested. All the extracts showed significant inhibition zone at 2500ppm against all bacterial strains. The highest inhibition was showed by ethanolic extract at 2500ppm against Xanthomonas, Clavibacter and Bacillus which were 16.25±0.353, 17.75±0.35 and 20.5±0.70 respectively. At 100ppm the extracts showed low inhibition zone against strains which were 10, 9.75±0.35, 11, respectively (Table 5, Figure 4).
In the current work ethanolic, methanolic, acetone, n-hexane and chloroform extracts of O. pes-caprae were examined for antifungal potential against fungalstrains likeAspergillus flavus, Penicillium and Fusarium solani at 100 and 1000ppm. The results are presented in Table 6 and Figure 5. Ageneral trend of dose dependency was observed i.e. effect became more pronounced withincreasing concentration of the various tested samples. Ethanolic extract was found effective againstA. flavus (48.24±1.24%) followed by Fusarium solani (41.5±0.70 %) and Penicilliun (35.34±0.98 %) at 1000ppm.
Many researchers conducted the activity on different plants like Ali et al.21 (2009) analyzed root extracts of Euphorbia wallichii for cytotoxicity. Nisar et al.22 (2011) reported crude methanolic extract and various fractions from Zizyphus oxyphylla stem. Khuda et al.23 (2012) investigated the extracts from leaves of X. strumarium, Achyranthes aspera, Valeriana wallichii and roots of Duchesnea indica for cytotoxic activity.Our study is in line with these workers.
Hameed et al.24 (2013) evaluated methanolic and acetone extracts of the Withaniasomnifera, Datura innoxia, Solanum surattense, Withania coagulans and Solanum nigrumfor phytotoxicity. Shah et al.25 (2013) demonstrated crude methanolic extract and solvent fractions of Conyza bonariensisfor the phytotoxicity. Rehmanullah et al.26 (2014) performed phytotoxic activity on Euphorbia helioscopa and Euphorbia hirta. These all workers have carried out similar studies which strengthen the present findings.
Various other workers have also conducted such studies on various plants, e.g., Hafeez et al.27 (2014) reported antibacterial activity of Eruca sativa, Bombax malabaricum, Chorisia speciosa,Bauhinia variegate, Kigelia Africana, Nigella sativa, Pinus halepensis, Zingiber officinale, Piper nigrum, Albizzia lebbeck, Ceiba pentandra, Origanum majorana, Rosmarinus officinalis, Ocimum basilicum, Thymus vulgaris, Mentha piperita, Simmondsia chinensis, Linum usitatissimum, Pimpinella anisum and Brachychiton opulneus. All extracts exhibited significant effect. He concluded that best aqueous extract was from A. lebbeck. Madureira et al.28 (2014) determined the antimicrobial potential of Sanguisorba hybrid. All extracts were tested againstyeast Candida albicans, Gram-negative (Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa,) Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, Mycobacterium smegmatis, Enteroccocus faecalis). Our study is in line with these workers.
Similar antifungal investigations were performed by several workers on various plants. Khan et al.29 (2011) worked out antifungal activity of Adhatoda vasica, Viburnum cotinifolium, Vitex negundo, Peganum harmala,Mimosa rubicaulis, Broussonetia papyrifera, Chenopodium ambrosoides, Euphorbia hirta, Taraxacum officinale, Urtica dioica, Verbascum Thapsus, Caryopteris grata and Woodfordia fruiticosa, Nisar et al.22 (2011) performed antifungal activity on crude methanolic extract and different fractions from Zizyphus oxyphylla stem against 5 fungal strainsviz., Aspergilus flavus, Fusarium solani, Microsporum canis, Candidaglaberataand Candida albicans. Shah et al.25 (2013) analyzedConyza bonariensisfor antifungal activity. Our study is in line with these workers.
CONCLUSION AND RECOMMENDATION
In O. pes-caprae highest significant cytotoxicity was shown by ethanolic extract at 1000ppm with LD50 8.98 which revealed this plant contains a good cytotoxic compound. The highest significant phytotoxicity was shown by chloroform extract at 1000ppm with FI50 0.1048 which depicted that this plant contains a natural weedicide character. The highest antibacterial activity was shown by ethanolic extract against Xanthomonas, Clavibacter machengnitisand Bacillus which was 16.25±0.353, 17.75±0.35, 20.5±0.70. The highest antifungal activity was shown by ethanolic extract against A. flavus (48.24±1.24%) followed by Fusarium solani (41.5±0.70 %) and Penicilliun (35.34±0.98 %) at 1000ppm. It can be concluded that this plant is a natural antibacterial and antifungal. Pharmacological potentials like cytotoxic, phytotoxic, antibacterial and antifungal activities of plants showed that these plants can be used in upcoming time for extracting numerous active constituents for their pharmacological preparations. These results might be exploited as cheaper effectual remedies for different ailments.
In present study Oxalis pes-caprae belonging to family oxalidaceae evaluated for pharmacological activities like cytotoxicity, phtotoxicity, antibacterial and antifungal bioassays. Its ethanolic extract showed highly significant (100%) cytotoxicity with LD50 8.98 at 1000 ppm. Lemna minor phytotoxicity asssay showed that O. pes-caprae chloroform extract showed 90% inhibition at 1000 ppm with FI50 0.1048. Antibacterial bioassays showed that all the samples of the plant were significant against Xanthomonas, Clavibacter machengnitis and Bacillus at 1000, 1500 and 2500 ppm doses. Dose dependent antifungal activities against test species (Aspergillus flavus, Penicillium and Fusarium solani) were noticed for all the extracts at 100 and 1000 ppm. Pharmacological potentials showed that this plant can be used in upcoming time for extracting numerous active constituents for their pharmacological preparations.
OPE: Oxalis pes-caprae ethanol; OPM: Oxalis pes-caprae methanol; OPA: Oxalis pes-capra eacetone; OPH: Oxalis pes-caprae n-hexane; OPC: Oxalis pes-caprae chloroform.
Cite this article: Naila S, Ibrar M. Pharmacological Studies of Oxalis Pes-caprae L. Pharmacog J. 2018;10(4):705-11.